Sphingosine 1-phosphate (S1P), a potent lipid mediator, acts through the G protein-coupled receptors to regulate vascular maturation, angiogenesis and immune cell trafficking. It is produced by the phosphorylation of sphingosine by the sphingosine kinase (Sphk)-1 and -2. Studies supported by this grant showed that Sphk isoenzymes are located at distinct subcellular compartments, Sphkla isoform is efficiently exported to the extracellular milieu as a functional kinase, a large S1P gradient is maintained between blood and interstitial compartments, Sphk2 is involved in the activation of the immunosuppressant drug FTY720, Sphkl is upregulated in the intestinal tumor tissues and that intracellular metabolism of sphingosine by Sphkl is critical for tumor cell proliferation in the ApcMin/+ model of intestinal neoplasia. These data support the emerging concept that Sphk isoenzymes function at different subcellular locations to precisely regulate the concentrations of sphingolipid mediators, thus establishing the S1P gradient between blood and interstitium. This proposal is based on the hypothesis that the function of Sphk isoforms is critical for the establishment of S1P gradient, receptor activation and the intracellular metabolism of sphingolipid mediators. Thus the specific aims are: 1. To determine the molecular basis for the establishment of the S1P gradient between blood and tissues. The hypothesis that extracellular export of Sphkl a isoform contributes to high plasma S1P levels will be tested in mouse models. Subcellular localization, activation-dependent translocation and receptor activation of Sphk will be characterized in live vascular cells using photoactivatable (PA)-GFP fusion proteins. 2. The S1P gradient is critical for receptor-dependent signaling in the vascular and immune cells. The cellular source of Sphk that contribute to high plasma levels will be determined. Whether high plasma S1P levels are required for optimal angiogenesis response will be tested in Sphk null mice. Contributions of hematopoietic and vascular cells in maintaining blood S1P levels and the angiogenic response will be investigated. 3. The Sphkl gene is essential for the proliferative expansion of adenomatous polyps in the ApcMin/+ model of intestinal neoplasia. We will test the hypothesis that Sphk enzymes regulate intracellular sphingosine levels, which directly affect the cell-cycle machinery to inhibit cell proliferation. These studies are anticipated to further elucidate the logic of S1P signaling system in pathophysiology. This information is especially warranted as the S1P analog FTY720-P, an immunomodulator and an inhibitor of vascular permeability is undergoing phase III clinical trials.